The activation of key cyclin-dependent kinases (cdk's) controls passage through regulatory points of the cell cycle known as transition points. As their name implies the activation of these kinases depends on the physical association of the catalytic cdk subunits with regulatory subunits known as cyclins. In addition there are a large number of other mechanisms that contribute to the regulation of these kinases. These mechanisms include both positive and negative regulatory phosphorylation events, protein turnover of the cyclins, interaction with kinase inhibitors, and mechanisms that control assembly of the subunits. For mammalian cells most of the key regulatory events that control commitment to cell division occur in the G1 phase of the division cycle. There are two G1 pathways in which cdk's participate. One of these pathways relies on the activation of cyclin D/cdk4 or cyclin D/cdk6 kinases. The activation of the cyclin D/cdk complexes is a key subject of studies in cell cycle control and oncogenesis. These kinases are activated in mid-G1 and the main phosphorylation target appears to be the retinoblastoma tumor suppressor protein (pRB). pRB is the prototype for the tumor suppressor gene products and is frequently mutation in human tumors. Other proteins in this pathway have also been shown to be mutated in tumors. Both cyclin D and cdk4 have been shown to act as oncogenes and their genes are frequently amplified in human tumors. An upstream regulator of cdk4 and cdk6 is a recently characterized kinase inhibitor known as p16. p16 is a tumor suppressor gene product in its own right and it is at least one of the tumor suppressor genes that predisposes individuals to hereditary melanoma. We have been studying the link between cell cycle regulatory events and oncogenesis. In the next granting cycle we propose to continue our studies on cyclin D/cdk control. Our first specific aim will be to determine how the function of the cyclin D/cdk inhibitor p16 is altered in human tumors. In preliminary work we have developed three assays to test p16 function. These assays will be used to determine the mechanism of cyclin D/cdk inhibition, to investigate the multifunctional activities of p16, and to study the cell type specificity of p16 inhibition. In our second aim we will determine the functional roles other members of the p16 family. There now are four members of the p16 family, and the roles of these inhibitors will be compared. The third aim will be to use existing and novel mutagenesis techniques to determine how specificity is achieved in the inhibitor/cdk interactions. Finally, we will study two mechanisms of cyclin D/cdk regulation that are still poorly understood. These are the serum stimulated assembly of cyclin D and cdk4 complexes and the cloning and characterization of a novel cyclin D/cdk6 associated protein.